Synthesis of Mycoplasma arginine deiminase in E. coli using stress-responsive proteins.

Abstract

We found Escherichia coli proteins, elongation factor Ts (Tsf), and malate dehydrogenase (Mdh) that can exist in the form of native and soluble proteins even under stress situation such as heat shock and protein denaturing condition. To examine their property as solubility enhancers, aggregation-prone Mycoplasma arginine deiminase (mADI), which has been suggested as anti-cancer agent, was fused to the C-terminal of each of them and cloned into pET28a to be expressed in the E. coli cytoplasm. When mADI was fused to fusion partners (Mdh, Tsf), a significant portion of the recombinant mADI was expressed in a soluble fraction (>90%) whereas the directly expressed mADI was aggregated to the inclusion body. In addition, recombinant mADI released from the fusion tag retained its soluble form and presented its specific enzymatic activity of converting l-arginine into citrulline (>10 U/mg). These results show that Tsf and Mdh could serve as effective solubility enhancers for aggregation-prone proteins (e.g. mADI in this case) when used as fusion expression partners in bacterial expression systems.

• Authors: Keum-Young Ahn, Boram Lee, Kyung-Yeon Han, Jong-Am Song, Doo Sung Lee, Jeewon Lee
• Journal: Enzyme and microbial technology (Enzyme Microb Technol) Vol. 63 Pg. 46-9 (Sep 2014) ISSN: 1879-0909 [Electronic] United States
• PMID: 25039059 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2014 Elsevier Inc. All rights reserved.

Chemical References

• Bacterial Proteins
• Escherichia coli Proteins
• Peptide Elongation Factors
• Recombinant Fusion Proteins
• elongation factor Ts
• Citrulline
• Arginine
• Malate Dehydrogenase
• Hydrolases
• arginine deiminase

Topics

• Arginine (metabolism)
• Bacterial Proteins (biosynthesis)
• Citrulline (metabolism)
• Colorimetry
• Electrophoresis, Polyacrylamide Gel
• Escherichia coli (genetics, metabolism)
• Escherichia coli Proteins (genetics, metabolism)
• Genes, Synthetic
• Genetic Vectors (genetics)
• Hydrolases (biosynthesis)
• Malate Dehydrogenase (genetics, metabolism)
• Mycoplasma (enzymology, genetics)
• Peptide Elongation Factors (genetics, metabolism)
• Recombinant Fusion Proteins (metabolism)
• Solubility